Host cell proteins (HCPs) are process-related impurities that are generated by the host organism, and are typically present at low levels in therapeutic monoclonal antibody (mAb) and other recombinant biopharmaceutical products. Firstly, a high-pH-low-pH "two-dimensional" reversed-phase nano-LC-MS/MS label-free quantification (2D nano-LC-MS/MS LFQ) method with a robust stability (CV% <
20%) was developed. Subsequently, this study developed economical hexamer ligand (HWRGWV) magnetic beads (HLMB), which aim to improve the sensitivity and reliability of HCP detection and has an IgG antibody-binding capacity similar to that of Protein A. In turn, the HCP study based on 2D nano-LC-MS/MS, a comparison between the removal and the reservation of the main antibody components was developed, and the qualitative and quantitative results were compared among HLMB/Protein A depletion and other two pretreatment processes. Results of this study indicated that after using HLMB/ProA material for antibody primary component removal, the content of HCPs in the elution buffer (associated or co-purified with the antibody) was significantly higher than that in the flow-through. In total, 22 kinds of HCPs were co-identified in HLMB eluent and ProA eluent, in which most of the HCPs exhibited weak alkalinity, while the sensitivity of HCPs identified with a low molecular weight (ranging from 13 to 21 kDa) was as low as 0.1 ppm. Together, this study established an economical and effective approach to comprehensively evaluate HCPs in antibodies, while also globally presenting the impact of major antibody components on the qualitative and quantitative analyses of HCPs.